Regulation of immune responses is central for the prevention of inflammatory and autoimmune disorders. While downregulation of the immune system can be achieved by way of immunosuppressive therapy, agents that generally suppress the immune system leave subjects susceptible to other disorders, including infections and cancers. A means for controlling the aberrant activation of an immune response to specific antigens would be a major advance in the treatment of autoimmune disorders, graft versus host disease and the side effects of gene therapy, as it would allow downregulation of the immune response against a particular target antigen, but would otherwise leave the immune system functional against invading pathogens and tumor associated antigens. Conversely, methods of specifically improving immunogenicity of specific antigens to which immune responses are desired would be of tremendous benefit in promoting desired immune responses; for example in the context of vaccination and promoting responsiveness to antigens including tumor antigens.
T helper (Th) cells are a class of CD4+ cells that function to regulate the proliferation of B cells and B cell responses. Th cells play an importance role in humoral immunity and immunopathology. Follicular helper T cells (TFH) are a recently defined subset of CD4+ T cells that are essential for helping cognate B cells form and maintain the germinal center (GC) reaction, and for development of humoral immune responses. These cells are universally defined by expression of the chemokine receptor CXCR5, which directs them to the B cell follicles via gradients of the chemokine CXCL131. TFH cells also express the transcription factor Bcl6 (which represses Blimp-1/Prdm1) and high levels of the costimulatory receptor ICOS, which are both critical for their differentiation and maintenance1-4. In addition, TFH cells secrete large amounts of IL-21, which aids in GC formation, isotype switching and plasma cell formation5. In humans and mice functionally similar TFH cells can be found in secondary lymphoid organs. CXCR5+ TFH cells are also present in peripheral blood and seen at elevated levels in individuals with autoantibodies, including systemic lupus erythematosis, myasthenia gravis and juvenile dermatomyositis patients. However, the function of these circulating TFH remains unclear6-9.
Regulatory T cells (Tregs) have pluripotent anti-inflammatory effects on multiple cell types. In particular, they control the activation of innate and adaptive immune cells. Tregs acting in an antigen-specific manner reduce effector T cell activation and function, for example, after effector T cells have successfully mounted an attack against an invading pathogen, or to suppress reactivity to self-antigen and thereby prevent autoimmune disease.
Two subsets of Tregs are classified according to the location at which they develop in vivo. Naturally occurring Tregs (nTreg) develop in the thymus and suppress self-reactive immune responses in the periphery, whereas adaptive Tregs (aTreg) develop in the periphery from conventional CD4+ T cells to ensure tolerance to harmless antigens, including those derived from, for example, food and intestinal flora. Both subsets of Treg cells are characterized by expression of high levels of CD25 and the transcription factor Foxp3. Tregs are thought to inhibit the antigen-specific expansion and/or activation of self-reactive effector T cells and to secrete suppressive cytokines, including TGF or IL-10. Because of their potential to provide antigen-specific immune regulation without generalized immunosuppression, Tregs have been contemplated for use in cell-based therapy for inflammatory or autoimmune disorders.
T follicular regulatory (TFR) cells are newly defined subset of CD4+CXCR5+ cells which are positive for the transcription factors FoxP3, Bcl6 and Prdm1/Blimp1 and function to inhibit the germinal center response21-23.
PD-1 has been identified as a receptor which binds to PD-L1 and PD-L2. PD-1 is a member of the immunoglobulin gene superfamily. PD-1 (Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994) Genomics 23:704; U.S. Pat. No. 5,698,520) has an extracellular region containing immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an immunoreceptor tyrosine-based inhibitory motif (ITIM). PD-1 transmits a negative signal to immune cells, similar to CTLA4. PD-1 ligand proteins are expressed on the surface of antigen presenting cells, and other cell types; and can provide a costimulatory signal to immune cells or can transmit downmodulatory signals to immune cells, depending upon the protein to which they bind. While transmission of an inhibitory signal leads to downmodulation in immune cell responses (and a resulting downmodulation in the overall immune response), the prevention of an inhibitory signal (e.g., by using a non-activating antibody against PD-1) in immune cells leads to upmodulation of immune cell responses (and a resulting upmodulation of an immune response).
TFH cells express high levels of programmed death (PD) 1 receptor (CD279). Signaling through PD-1 attenuates TCR signals and inhibits T cell expansion, cytokine production and cytolytic function. In addition, PD-1 promotes the development of induced regulatory T (iTreg) cells from naïve lymphocytes10-14. PD-1 has two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). PD-L1 is more widely expressed than PD-L2, but PD-L1 and PD-L2 both can be expressed on GC B cells and dendritic cells15. Perturbation studies suggest critical roles for this pathway in regulating humoral immune responses. However, there are conflicting reports as to the function of the PD-1 pathway in controlling humoral immunity. Some studies have found that humoral responses are attenuated16-18, while others have seen that humoral responses are heightened19, 20 when PD-1:PD-L interactions are prevented.
PD-1 also is found on TFR cells. These cells originate from natural regulatory T cell precursors, but express similar levels of ICOS, CXCR5 and PD-1 as TFH cells. Since ICOS, CXCR5 and PD-1 have been widely used to identify and purify ‘TFH cells’, it seems likely that the inability to define clear functions for PD-1 in GC responses derives from experimental systems containing mixtures of stimulatory TFH cells and inhibitory TFR cells.
The present inventors have discovered that PD-1:PD-L1 interactions limit TFR cell differentiation and function. This discovery has elucidated novel approaches to modulating an immune response for use in therapy.
This discovery has also elucidated novel cell markers for identifying and separating TFR and TFH cells from all other cell types. These markers are also useful for selectively modifying TFR-mediated and/or TFH mediated immune responses in vivo and in vitro.